Menadione also inhibited the cell growth in A549 cells (IC50: 16 µM). Quantitative RT-PCR showed significant downregulation of CSC markers  and Snail, epithelial-mesenchymal transition marker, after treatment with menadione and cisplatin/gemcitabine. Flow cytometry showed CD44-positive cells that constitute a high percentage (70%) of A549 cells reduced significantly after treatment with cisplatin/gemcitabine or menadione. However, A549 cells did not show a significant population positive for CD133 and ABCB1 (less than 0.05%), and these fractions did not change after treatment with two agents.Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Chemotherapy-induced adverse effects and resistance of NSCLC to conventional drugs reduce the efficacy of current therapies. Tumors contain a small population of cancer stem cells (CSCs) that play a critical role in tumor initiation, maintenance, and drug resistance that finally lead to cancer recurrence. Therefore, CSC-targeting therapies can offer the best hope for developing curative cancer therapies. Vitamins have a high potential for cancer prevention and treatment. Vitamins also ameliorate the side effects which occur in chemo-radio therapy. Menadione  is a synthetic form of vitamin K that indicated antitumor activities. The purpose of this study was to evaluate the anti-CSCs effect of menadione and combination of cisplatin and gemcitabine as a first-line treatment in patients with NSCLC on the NSCLC cell line A549. MTT results displayed decreased cell survival after treatment with cisplatin/gemcitabine for 48 h treatment (IC Lung cancer is one of the most commonly occurring cancers and the leading cause of cancer-associated death worldwide . Non-smaVitamins are compounds required for normal physiological function and growth of the body. Vitamins are reported to have an apoptotic and inhibitory effect against various cancers. Therefore, the role of vitamins in cancer prevention and treatment has emerged in the past few decades. Furthermore, it is reported that vitamins involve in the amelioration of side effects that occur in chemotherapy and radiation therapy -31. As aEMT is a process by which immotile epithelial cells lose cell-cell adhesion and obtain migratory and invasive properties, which have been shown to occur during metastasis in cancer progression , 40. SigMaterialsPlatinol) and gemcitabine (Gemzar) were purchased from Vianex S.A, Greece and Sobhan, Iran, respectively, and menadione (Cat. No. M5625) was from Sigma-Aldrich. FITC anti-human CD44 antibody , PE anti- Human CD133/2 , PE anti-Human CD243 (ABCB1) antibody , Propidium iodide (PI)  and 7-amino-actinomycin D (7-AAD)  were used for flow cytometry. Total RNA isolation kit was purchased from DENAzist Asia, Mashhad, Iran. RNase-free DNase I (Cat. No. EN0521) and M-MuLV reverse transcriptase (Cat. No. EP0441) was from Thermo Scientific, Wilmington, USA. The real-time PCR was performed using Real QPCR 2x SYBR Green master mix . Oligo(dT)18 primer and dNTP were obtained from Yekta Tajhiz Azma, Tehran, Iran. We prepared the RPMI-1640 medium from Gibco, Grand Island, USA, and fetal bovine serum (FBS) and penicillin-streptomycin from Biowest, France. 3--2, 5-diphenyltetrazolium bromide (MTT) was from Atocel, Austria. For cell treatment, cisplatin /gemcitabine (5 µM) and 16 µM menadione separately. RNA isolation, quantitative reverse transcription PCR (qRT-PCR) and flow cytometry analysis was carried out on untreated and treated cells to evaluate the gene expression level.Human lung cancer cell line A549 was obtained from the National Cell Bank of Iran . They were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained at 37 °C in humidified air with 5% COMeasurement of cell viabilityin-vitro cytotoxicity of gemcitabine and cisplatin as single agents and their combinations as well as menadione on the A549 cell line. The cells were seeded in 96-plates at a density of 5 × 103 cells/well and allowed to adhere overnight. Cells were exposed to different concentrations of each drug as shown in 50 values of agents were calculated using Prism 6.0 .MTT  assay was conducted to evaluate Quantitative reverse-transcription PCR (qRT-PCR)Total RNA was extracted using an RNA isolation kit according to the manufacturer’s instructions and was reverse-transcribed by using M-MuLV reverse transcriptase as described in the protocol. Oct4, Nanog, Aldh1a1, Abcb1, CD133, CD44, Gapdh and Snail were: 95 ºC for 15 min, followed by 40 cycles of 95 ºC for 20 sec, 64 ºC for 30 sec, and 72 ºC for 15 sec. The same program was used for Sox2, except that the annealing temperature was 66 ºC. At the end of the PCR runs to derive melting curves, the temperature was increased in steps of 1 ºC for 5 sec from 60 ºC to 95 ºC. Analysis of melting curves clearly indicated that each of the primer pairs amplified a single expected product with a distinct Tm. The accuracy of the amplification reaction was validated by gel electrophoresis. Gapdh was used as an endogenous control to normalize each sample. Relative quantification of mRNA within the samples was examined using the comparative Ct method . Real-time PCR was carried out on Thermocycler . Each reaction consists of 1X SYBR Green Real-time PCR Master Mix, 1 µL cDNA template, and each primer at 250 nM in a 20 µL reaction volume. Gene-specific primers were designed using Oligo7 Primer Analysis Software. The sequence of primers and product length are described in Table 1. Amplification conditions for Flow cytometryThe following protocol was employed to examine the expression of cell surface markers CD44, CD133, and ABCB1 by flow cytometry. Cells were dissociated with trypsin-EDTA and after washing, one million cells were suspended in PBS/2% FBS. The antibodies were added to cells at a 1:5 dilution for FITC-CD44 antibody and 1:10 dilution for PE-CD133 and PE-ABCB1 antibodies for 30 min in the dark. Finally, after washing, cells were suspended in 0.5 mL of PBS/2% FBS and then analyzed by flow cytometry. To detect dead cells, PI was used with FITC-CD44 antibody and 7-AAD was used with PE-CD133 and PE-ABCB1 antibodies. Flow cytometry was done using a FACSCalibur . Data were analyzed by Flow Jo software, which is provided with the system.Statistical analysist-test, and P < 0.05 was considered to indicate a statistically significant difference. All the experiments were repeated for 3 times.All data were expressed as Mean±SD and analyzed using SPSS 16.0 software . Data analysis was performed using Student’s Dose-dependent antitumor activity of chemotherapy drugs and menadione against NSCLC cell lines A54950 values were 22.5 µM for cisplatin , 47Oct4,FACS analysis of CSC surface markers following cisplatin/gemcitabine and menadione treatment in A549 cellsCD133 and CD44 are recognized as robust markers for CSCs in solid tumors -50. TherPlatinum-based doublet chemotherapy is the gold standard therapy for NSCLC patients where cisplatin or carboplatin are used in combination with drugs such as paclitaxel, gemcitabine, or vinorelbine . DespiteOct4, Nanog, and Sox2 show that this vitamin can inhibit self-renewal ability and induce differentiation of CSCs in the A549 cell line. The combination of cisplatin and gemcitabine which is used as a first-line regimen for patients with advanced NSCLC, also downregulated the expression of pluripotent markers. In other studies, downregulation of stem cell-associated genes was indicated after treatment with some chemotherapy drugs such as FOLFOX , docetaxel, and gemcitabine as well as phytochemicals agents such as berberine, apigenin, cinnamic acid, and kaempferol  reduced the expression of CSCs markers in NSCLC cell line A549. OCT4, NANOG, and SOX2 are three essential transcription factors contributing to pluripotency and self-renewal of normal and CSCs , 11. Menempferol -58. CD44 and CD133 mRNA expression. As a result, it seems that these components can reduce proliferation, tumorigenesis, and stem cell-like properties in A549 cancer cells. Expression of cell surface markers CD44 was detectable on around 70% of the A549 cell line and this ratio decreased significantly after treatment with menadione and cisplatin/gemcitabine that is consistent with qRT-PCR results. However, using our antibody, we were not able to detect CD133 expression on a significant proportion of A549 cells as well as menadione or cisplatin/gemcitabine-treated cells. Moreover, CSCs are identified and isolated from various tumors based on overexpression of specific surface markers . CD44 anAldh1a1. Overexpression of the ABC gene family is one of the principal mechanisms for protecting CSCs against many cytotoxic drugs that contribute to cancer resistance to chemotherapy (Abcb1 in cisplatin/gemcitabine and menadione-treated cells. Moreover, flow cytometry did not show detectable expression of ABCB1 on A549 cells, and this proportion did not change after treatment with two agents. Stem cell populations in many types of tumors are defined by the high activity of ALDHs. ALDHs are a superfamily of enzymes responsible for oxidizing endogenous and exogenous aldehydes to carboxylic acids. Therefore, ALDHs activity confers CSC resistance to antitumor drugs, so components that can reduce the expression of this marker or inhibit its activity play a key role in sensitizing resistant cells to cytotoxic agents -69. The otherapy . Drug-meotherapy . In anototherapy , 72. OurAs a key transcriptional repressor of E-cadherin, Snail is recognized as a prominent inducer of EMT and tumor metastatic , 74. ConThe promising role of vitamins, mostly vitamin A and D, in cancer prevention and treatment has emerged from the past few decades . FurtherIn-vivo experiment is needed to achieve more reliable results on the anti-CSCs effect of menadione and further validate the applicability of our results.In conclusion, tumorigenic ability and resistance of CSCs to conventional chemo- and radio-therapy make them a potential target for anticancer drugs to reduce drug resistance and attenuate the likelihood of relapse. Here we introduced menadione as an anticancer agent that can down-regulate the expression of important CSC markers. Although treatment with cisplatin and gemcitabine showed approximately the same effect as menadione on downregulation of CSC-associated genes, combination therapy with menadione and chemotherapy drugs may not only reduce adverse side effects but improve the effectiveness of chemotherapy drugs and finally achieve prolonged survival of cancer patients.


Ganoderma tsugae  for 25 weeks to investigate the effects of GTDE on oxidative stress and memory deficits in the D-galactose-induced aging rats. We found that GTDE significantly improved the locomotion and spatial memory and learning in the aging rats. GTDE alleviated the aging-induced reduction of dendritic branching in neurons of the hippocampus and cerebral cortex. Immunoblotting revealed a significant increase in the protein expression levels of the superoxide dismutase-1 (SOD-1) and catalase, and the brain-derived neurotrophic factor (BDNF) in rats that received GTDE. D-gal-induced increase in the lipid peroxidation product 4-hydroxynonenal (4-HNE) was significantly attenuated after the administration of GTDE, and pyrin domain-containing 3 protein (NLRP3) revealed a significant decrease in NLRP3 expression after GTDE administration. Lastly, GTDE significantly reduced the advanced glycosylation end products (AGEs). In conclusion, GTDE increases antioxidant capacity and BDNF expression of the brain, protects the dendritic structure of neurons, and reduces aging-induced neuronal damage, thereby attenuating cognitive impairment caused by aging.Lingzhi has long been regarded as having life-prolonging effects. Research in recent years has also reported that Lingzhi possesses anti-tumor, anti-inflammatory, immunomodulatory, hepatoprotective, and anti-lipogenic effects. The D-galactose -induced aging Long-Evans rats were simultaneously orally administered a DMSO extract of  Aging, a slow and progressive process of physiological change, is an inevitable natural phenomenon that is irreversible. The rate at which aging occurs is affected by complex interactions among genetics, the environment, diet, and lifestyle habits. Aging is often accompanied by a decline in physiological function, which decreases activities of daily living (ADL) and, consequently, increases healthcare and social resource demands , 2. SeveD-galactose  is a reducing sugar that produces ROS when it is metabolized in the body . It inteGanoderma tsugae), a type of Lingzhi widely used in Asia, was selected for the treatment of D-gal-induced aging rats. The hydroxyl radical-scavenging effect and metal ion chelating ability of G. tsugae give rise to superior antioxidant effects [Ganoderma tsugae   for 25 weeks. We aimed to investigate whether the administration of G. tsugae attenuated oxidative stress and memory deficits in D-gal-induced aging rats.Lingzhi  is an edible medicinal mushroom that has been regarded as a herb with health-promoting and life-prolonging effects in East Asian countries for several centuries . It cont effects , 32. Morp = 0.9357, F = 0.1389; Brain weight: p = 0,3334, F = 1.199). The values of the physiological parameters for the D-gal+GTDE group were similar to those for the control group. Compared with the control group, the D-gal group had a slightly lower hemoglobin concentration, slightly higher red blood cell (RBC) count, and decreased white blood cell (WBC) count . In aging rats fed with GTDE, a trend of recovery towards the values of the control group was observed in these parameters, but the differences in the various physiological parameters were not statistically significant. These experimental results indicate that injections of D-gal and the oral administration of GTDE for 25 weeks did not affect the basic physiological parameters of the rats.We first measured the basic physiological parameters of the various groups to evaluate the effects of GTDE on the rats. As shown in p < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 3.636, p = 0.0328) indicate that the frequency with which the rats entered the central zone was highest in the D-gal group compared with the other groups, whereas the frequency was significantly lower in the D-gal+GTDE group than in the D-gal group.The open-field test is often used for the evaluation of behaviors or emotions such as locomotion, exploration, and anxiety by taking advantage of the thigmotactic behavior of animals , 34. In p < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 5.158, p = 0.0075; p < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 5.442, p = 0.0059).Both the familiar/novel object recognition test and familiar/novel location recognition test are methods for high-level verification of recognition memory . The novp < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 4.976, p = 0.0034). The analysis shows that the D-gal group was less likely to reach the platform than the control group. By contrast, the likelihood of the D-gal+GTDE group reaching the platform was greater, which was similar to that of the control group. These results indicate that the aging rats suffered from age-associated spatial memory impairment, which was alleviated with the administration of GTDE.The Morris water maze is used to assess spatial learning and memory in animals . At the p = 0.0893 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 1.842).Besides its use for the classical spatial long-term memory test, the Morris water maze can also be used to assess working memory, which represents the ability to learn new things, by moving the platform to a new location each day , 38. Thep > 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, 108 μm from the soma: F = 2.367, p = 0.0786; 120 μm from the soma: F = 2.559, p = 0.0623). p > 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, branch points: F = 1.442, p = 2.2384; terminal points: F = 1.149, p = 0.3359); (2) Sholl analysis of the neurons of the hippocampal CA3 region of the various groups showed fewer intersections in the D-gal group than in the control group—the difference was statistically significant within distances of 25–50 μm from the soma . They were significantly more branch points in the D-gal+GTDE and D-gal groups within distances of 20–108 μm ; (3) Sholl analysis of the neurons of the hippocampal dentate gyrus region of the various groups showed that the D-gal group had fewer intersections than the control group, but the difference was not statistically significant . The dendritic branch points and terminal points were fewer in the D-gal group than in the control group, but the differences were not statistically significant. The D-gal+GTDE group had more dendritic branch points and terminal points than the D-gal group; the difference in the number of dendritic terminal points was statistically significant . These demonstrated that the administration of GTDE attenuated the aging-induced decrease in dendritic branching in certain hippocampal regions of aging rats.The complexity of the dendritic branching determines whether a good synaptic function exists in neurons and affects their ability to process and consolidate information . Fig 2a 0–108 μm . The counificant . Comparep < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, 60 μm from the soma: F = 8.863, p<0.0001; 96 μm from the soma: F = 9.264, p<0.0001). From p < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, branch points: F = 8.440, p<0.0001; terminal points: F = 8.599, p<0.0001). The results above indicate that GTDE attenuated the aging-induced decrease in dendritic branching in the cerebral cortex of aging rats.The results of Sholl analysis of the neurons in the cerebral cortex indicate that the D-gal group had fewer intersections than the control group, with the difference being statistically different within distances of 60–162 μm . Comparep < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 59.794, p<0.0001).To determine if GTDE alleviated the effects of aging on neuronal morphology and function through its antioxidant activity, we assessed the concentration of the lipid peroxidation product 4-hydroxynonenal (4-HNE) in the cerebral cortex. When oxidative stress causes damage in the body, 4-HNE is produced from the oxidation of polyunsaturated fatty acids, which makes it a suitable marker of oxidative damage . Fig 3a p < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, SOD: F = 58.586, p<0.0001; Catalase: F = 24.958, p<0.0001) and hippocampus . The expression levels of antioxidant proteins were measured to validate the theory that GTDE attenuated the effects of aging through its antioxidant activity. Previous research has reported a decrease in the activities of antioxidant enzymes in the body during the aging process . SOD-1 apocampus  . Moreover, we found that the NLRP3 expression inside each cell increased in the D-gal induced group compared to control, which could be attenuated by GTDE administration . The NLRP3 protein expression level in the cerebral cortical tissue of the D-gal group was 0.2 times higher than that of the control group whereas the expression level of the D-gal+GTDE group was 0.21 times lower than that of the D-gal group, with both differences in expression level being statistically significant . The NLRP3 protein expression levels in both the hippocampus and cerebral cortex of the GTDE group were lower than those of the control group, but the differences were not statistically significant.Several studies have shown that aging induces oxidative stress and causes an increased inflammatory response –43. Therp < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons test, F = 39.530, p<0.0001).Researchers have reported that aging usually causes an increase in the formation of AGEs , 45. AGEp < 0.05 using one-way ANOVA followed by the Tukey’s multiple comparisons tests, Cortex: F = 39.069, p<0.0001; Hippocampus: F = 604.00, p<0.0001). These results demonstrate that GTDE may promote or maintain BDNF expression in the hippocampus and cerebral cortex, which is beneficial to the maintenance of neuronal health.BDNF promotes neuron survival and differentiation and induces synaptic plasticity. In aging brains, BDNF expression generally demonstrates a decreasing trend, which is indicative of a reduction in neurogenesis and neuronal activity , 48. We Ganoderma lucidum) is a common species of the traditional Chinese herb, Lingzhi, and it is also commonly used in research. G. lucidum has mainly been used in clinical studies for cancer treatment [Ganoderma tsugae, another species of Lingzhi, which has better antioxidant effects than G. lucidum [G. tsugae attenuates aging-induced cognitive decline and protects neuronal morphology against aging through mechanisms such as antioxidant activity, reduction of inflammation, and anti-aging effects. As the dose of G. tsugae used in this study is 1/1000 of the no-observed-adverse-effect level [G. tsugae  to culture and collected the mycelium. He further extracted the G. tsugae from the fruiting body using hot water as described in the previously published work [G. tsugae was followed by DMSO precipitation, reserve dialysis, and protein depletion. The final content of triterpenoids is 128 g/100g, and the content of water-soluble polysaccharides is 0.39 g/100g in G. tsugae—DMSO solution. GTDE dosage (200 μg/kg/day) is followed by our previous preliminary study [The hed work . In briehed work . The driry study .Male Long-Evans rats (LE rats) (680–730 g) that were 24 weeks old  were used. The Institutional Animal Care and Use Committee of the Chung Yuan Christian University approved the use of animals in this study (Approved protocol no# 103019). They were kept under the following conditions: temperature of 18–26°C, the humidity of 30–70%, and a 12:12 h light-dark cycle with free access to laboratory chow and water throughout the study. The twenty-eight rats were randomly divided into four groups:  (n = 7 each group). In the D-gal treatment group, D-gal (100 mg/kg in PBS) was injected subcutaneously, and phosphate-buffered saline (PBS) in the same volume was given orally daily into the LE rats for 25 weeks. In the D-gal plus GTDE treatment group, GTDE  was administered as oral gavage daily concomitantly with D-gal injections for 25 weeks. In the GTDE treatment group, oral gavage of GTDE and subcutaneous injection of PBS in the same volume was performed daily for 25 weeks. All control animals were administered PBS in the same volume subcutaneously and orally. The dosage for D-gal and GTDE references the previous literature  and our An acrylic box measuring approximately 100×100×30 cm was divided by gridlines into nine equally sized squares that served as nine zones. Each rat was placed individually in the central square. Crossing was defined as the exiting of the rat from the previous square with all four paws. Between tests, the acrylic box was wiped with 75% alcohol to avoid odor interference. The frequency of crossing and duration spent in the center square within five minutes was recorded for each rat. Behavioral data were acquired by video recording and software analysis .NOR and NLR tests were used to assess the spatial hippocampal-dependent memory of the rats. The experiment was performed based on previously described methods , 79: (1)Morris water maze has been used to evaluate sensorimotor and memory deficits in several publications , 81. TheWorking memory refers to the part of short-term memory that involves storing and manipulating information for a relatively short period . The exp® 20 detergent (TBST). The blots were visualized by enhanced chemiluminescence (Promega™ ECL Western Blotting Substrate) and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences) . The total intensity of bands was analyzed by ImageJ . Briefly, an area with the same size as the selected region close to the bands was used to account for background intensity. The difference between the background and the band’s integrated density was determined to be the absolute intensity of the band. Equal protein loading was confirmed using GAPDH immunoreactivity. Quantification of protein expression was normalized and presented as a fold of the control. The lysate of each brain was randomly selected. Individual homogenates were run repeated at least three times. The experimenter was blinded to the condition.Brains were removed and dissected in PBS to obtain the hippocampus and cortex. The tissues were homogenized and lysed. Bradford assays were used to determine the protein concentrations of the extracts. The lysates were prepared in SDS containing sample buffer. Equal volumes with the same concentration of the lysates were separated by 10% or 12% SDS-PAGE gel using 50 μg protein per lane. The proteins were transferred to PVDF membranes using the electrophoretic transfer . The nonspecific reactivity of the membranes was blocked. The primary antibodies were rabbit anti-SOD-1 , rabbit anti-catalase , rabbit anti-BDNF , rabbit anti-NLRP3 , and anti-GAPDH  . The secondary antibody was diluted at 1:5000 in Tris-buffered saline with 0.1% Tween® 594-conjugated AffiniPure Goat Anti-Mouse IgG and Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG , mounted using a DAPI  -containing mounting medium, and observed under a fluorescent microscope  at 200x magnification. For comparing groups, the same setting was applied in all immunostaining images, including the brightness of the excitation light and the camera exposure time. The experimenter was blinded to the condition when taking images and analyzing. Images were processed and analyzed using ImageJ. For 4-HNE, the images were thresholded with the ROI below 80 and over 200. The intensity of each image was divided by the number of DAPI positive cells. The value of each group was then normalized to control. For NLRP3, the NLRP staining positive cells were calculated and divided by the number of DAPI positive cells. The value of each group was then normalized to control.After heart perfusion, the brain tissue of each rat was removed, immersed in 4% paraformaldehyde for 24 h, allowed to sink in 30% sucrose/PBS, embedded using the optimal cutting temperature (OCT) compound , frozen in liquid nitrogen, and sectioned into 10 μm slices. The samples were air-dried overnight, the nonspecific signals were blocked, and rabbit anti-4-HNE  and rabbit anti-NLRP3  were added. Subsequently, the sections were incubated with Alexa Fluro®-Multi+ Microplate Reader.The concentrations of AGEs were measured using the commercially available MyBioSource AGEs ELISA Kit following the manufacturer’s instructions. First, serum or plasma was diluted 20-fold using a sample diluent. The first antibody to detect AGEs is conjugated with biotin, and the second antibody is HRP-avidin. TMB  substrate was added to each well for reaction away from light at 37°C for 15–30 min. The reaction in each well was stopped by adding 50 μl of a stop solution followed by gentle plate shaking, and a color change from blue to yellow was observed after the reaction with the TMB Substrate. Absorbance at 490 nm was measured using a GloMax™ Kit according to the manufacturer’s instructions. Briefly, all the experimental steps were performed away from light. Approximately 1 cm3 of brain tissue was stained with the Golgi solutions, then embedded in a tissue freezing medium (Leica) and frozen with liquid nitrogen. The tissue was sectioned into 80 μm slices using a microtome at an operating temperature of -25°C. After being wet twice (4 min each) with deionized distilled water, the brain tissue was sequentially immersed in 50%, 75%, and 95% ethanol for 4 min each, four times in 100% ethanol (4 min each), and twice in xylene (2 min each). Lastly, the tissue sample was mounted with a mounting medium, and the CA1 region, CA3 region, and dentate gyrus of the hippocampus were observed under an optical microscope at 400x magnification. The experimenter was blinded to the condition when taking images.Golgi staining was performed using the commercially available FD Rapid GolgiStainSholl analysis was performed using the Bonfire program, a semi-automated tool for the analysis of dendrite morphology , 84. TheP < 0.05) was determined using one-way analysis of variance followed by post hoc Tukey’s Multiple Comparisons Test for multiple comparisons using GraphPad InStat software 3.05 package for Windows . F- and p- values were included.Data are presented as the mean ± standard error. Statistical significance (S1 File(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Background: The management of septic patients hospitalized in Internal Medicine wards represents a challenge due to their complexity and heterogeneity, and a high mortality rate. Among the available prognostic tools, procalcitonin (PCT) is considered a marker of bacterial infection. Furthermore, an association between vitamin D deficiency and poor sepsis-related outcomes has been described. Objectives: To evaluate the prognostic accuracy of two consecutive PCT determinations (Delta-PCT) and of vitamin D levels in predicting mortality in a population of patients with microbiological identified sepsis admitted to Internal Medicine wards. Methods: This is a sub-analysis of a previous prospective study. A total of 80 patients had at least two available consecutive PCT determinations, while 63 had also vitamin D. Delta-PCT was defined as a reduction of PCT > 50% after 48 h, >75% after 72 h, and >85% after 96 h. Mortality rate at 28- and 90-days were considered as main outcome. Results: Mortality rate was 18.7% at 28-days and 30.0% at 90-days. Baseline PCT levels did not differ between survived and deceased patients . A significantly higher proportion of survived patients showed Delta-PCT . Delta-PCT was associated with a lower 28-days  and 90-days mortality . A significantly higher proportion of deceased patients showed severe vitamin D deficiency . Severe vitamin D deficiency was associated with a higher 28-days  and 90-days mortality . Conclusions: Delta-PCT and vitamin D represent two useful tests for predicting prognosis of septic patients admitted to Internal Medicine wards. Sepsis is a leading cause of death and health care systems major burden worldwide . Sepsis Medical tools available for the management of septic patients, and used in daily clinical practice, have been mainly developed in Intensive Care Units (ICUs) and have not been extensively validated in Internal Medicine (IM) wards . Among tProcalcitonin (PCT) is a polypeptide released after the interaction between cytokine-activated macrophages and endothelial cells in response to bacterial components, particularly lipopolysaccharide . CirculaVitamin D is a hormone playing its primary role in the bone homeostasis, but it is also involved in regulating immunity, both innate and adaptive . The preThe aim of the present study was to evaluate the accuracy of two consecutive PCT determinations (Delta-PCT) and vitamin D levels in predicting mortality, among a population of IM inpatients with microbiological identified sepsis.The Internal Medicine Sepsis Study Group has promoted a 12-months sepsis surveillance program in two Internal Medicine Units of the “Agostino Gemelli” University Hospital, Catholic University of Rome, Rome, Italy . Sepsis This is a sub-analysis of a database including prospectively collected data of a cohort of consecutive patients with microbiological-identified sepsis admitted to an IM Unit . The stuData regarding 28- and 90-days mortality were retained from the initial study .PCT was assessed at the time of collection of blood cultures  and during antimicrobial treatment . As previously reported, a PCT level > 2 ng/mL was considered a significant cut-off for sepsis. Delta-PCT was calculated as the percentual variation of PCT at T1 compared to T0. We defined Delta-PCT as a reduction of PCT levels > 50% after 48 h or >75% after 72 h or >85% after 96 h from T0. Thus, a reduction of PCT levels lower than these cut-offs or a baseline PCT < 2 ng/mL was considered as “absence of Delta-PCT.”Vitamin D assay was available in 63 out 80 (78.7%) patients. Although the Endocrine Society suggests specific categories for different vitamin D levels D ≤ 19.9 ng/mL; vitamin D insufficiency: 20–29.9 ng/mL; vitamin D normal group: ≥30 ng/mL) ,20,21. A number of statistical procedures were applied for analysis of data, both descriptive and inferential, including numerical summaries (reported throughout the paper), Wilcoxon tests, Chi-square tests of independence, and logistic regression. The ultimate goal of the analysis was to primarily study the effect of Delta-PCT on 28-days and, respectively, 90-days mortality in a logistic regression framework. Moreover, vitamin D deficiency was studied in a similar way. First, a two-sample Wilcoxon test was run for assessing location differences of PCT levels at T0 between the group of deceased versus that of non-deceased patients. Then, a standard chi-squared test of independence in a two-way contingency table was used for tentatively testing the influence of both Delta-PCT and vitamin D deficiency on mortality. Afterwards, logistic regression was performed for an in-depth study of the effect of Delta-PCT as well as vitamin D deficiency on mortality. Obviously, all these analyses were repeated for both 28- and 90-days mortality. For the correct application of logistic regression, standard model checking techniques were run to assess model adequacy and thus validate the analysis method. All the computations were carried out by using the free software R . Main characteristics of the studied population have already been described  and are A total of 15 out 80 patients (18.7%) died at 28-days and 27 out 80 (30.0%) died at 90-days. Baseline PCT levels did not differ between survived and deceased patients. A total of 39 patients (48.7%) showed Delta-PCT. A total of 31 out 63 patients (49.2%) patients showed severe vitamin D deficiency. Vitamin D levels were significantly lower in deceased than survived patients.p = 0.525) nor at 90-days . The Chi-square tests of independence showed a significantly higher proportion of patients with Delta-PCT among survivors at 28-days (p = 0.002) (p < 0.001)  and 90-days mortality .The two-sample Wilcoxon tests showed that location values  of PCT levels at T0 did not differ between survived and deceased patients at 28-days   and at 9< 0.001) . The accp = 0.047) (p = 0.049)  and 90-days mortality , even if the level of significance was borderline.The Chi-squared tests of independence showed a higher proportion of patients with severe vitamin D deficiency among deceased patients at 28-days (= 0.047)  and at 9= 0.049) . The accp = 0.012, OR = 0.06, 95%CI 0.003–0.38) and 90-days  and a positive effect of severe vitamin D deficiency on mortality both at 28-days  and 90-days .Finally, a two factors logistic regression confirmed previous results in terms of a negative effect of Delta-PCT on mortality both at 28-days  in the IM setting have been evaluated by a few studies. To the best of our knowledge, the study by Pieralli and colleagues is the only one conducted in a non-ICU setting, aiming to evaluate the role of PCT kinetics in predicting 30-days mortality in a sample of 144 patients with severe sepsis and/or septic shock admitted to Emergency Departments (EDs) or general wards . As mainThe ability of Delta-PCT to be a better prognostic marker than single PCT measurement has several explanations. First, the value of a single PCT measurement as a predictor of outcome is poor given the large overlap between false negative and false positive values, different normal ranges and high interindividual variability due to acute comorbid states ,25. MoreIn the present sample of IM septic patients, the accuracy of Delta-PCT was better in terms of specificity/PPV, than sensitivity/NPV. In other words, it is a biomarker with better “rule-out” than “rule-in” performances. The present study confirms our previous observation on severe vitamin D deficiency as independent predictor of death. This observation is in line with a recent meta-analysis, that showed that lower vitamin D at admission was independently associated with increased risk or mortality in patients with sepsis, even applying different diagnostic criteria for sepsis  . HoweverLimitations of the present study are represented by the small sample size and the monocentric IM population. Moreover, results of our analysis have not been adjusted for potential confounders , given their lack of influence observed in the main paper . In any Sepsis is increasingly diagnosed in Internal Medicine wards. The management of septic patients with multiple comorbidities represents a real challenge due to the complexity of the syndrome and the high heterogeneity of septic populations. Even few and relatively easy assessments of biomarkers can be of help for patients’ outcome in certain conditions. Within this context, Delta-PCT and vitamin D could play a promising role for predicting the prognosis of septic patients admitted to Internal Medicine wards.


BN rats fed a low or high salt diet. We observed that 8 of 27 amino acids responded to a high salt diet in SS rats. Thus, we hypothesized that a defect in amino acids may contribute to the development of salt-induced hypertension. L-phenylalanine was used to treat SS rats with a low or high salt diet. The results demonstrated that L-phenylalanine supplementation significantly enhanced the serum nitrite levels and attenuated the high salt-induced hypertension in SS rats. Low levels of BH4 and nitrite and the impaired vascular response to acetylcholine were rescued by L-phenylalanine supplementation. Moreover, increased GTP cyclohydrolase (GCH1) mRNA, levels of BH4 and nitrite, and reduced superoxide production were observed in the kidneys of hypertensive SS rats with L-phenylalanine. The antihypertensive effects of L-phenylalanine might be mediated by enhancing BH4 biosynthesis and decreasing superoxide production from NO synthase, thereby protecting vascular and kidney function with reduced ROS and elevated NO levels. The present study demonstrated that L-phenylalanine supplementation restored vascular function, suggesting L-phenylalanine represented a potential target to attenuate high salt-sensitive hypertension through GCH1-BH4.Amino acid metabolism plays an important role in controlling blood pressure by regulating the production of NO and ROS. The present study examined amino acid levels in the serum of Dahl SS rats and SS.13 High salt-induced hypertension and cardiovascular diseases are important risk factors for death worldwide , 2. AlthIncreasing evidence has indicated that high salt-induced hypertension is a metabolic syndrome –7, and tBN rats [Our previous studies demonstrated that the metabolic features, especially amino acid metabolism, were significantly different between SS rats and SS.13BN rats . In addiBN rats , 16. TheBN rats . MoreoveBN rats –20. Howe4) production, as a cofactor for aromatic amino acid hydroxylation, involved in the relaxation of the endothelium [L-phenylalanine (L-phe), as the essential amino acid, is converted to tyrosine, and effects on blood pressure are potentially due to the changes in tyrosine levels by modulate the production of DOPA  and dopamine . Besidesothelium . However4 biosynthesis, would attenuate the increased blood pressure in high salt-induced Dahl SS rats.We hypothesize that amino acids metabolism may contribute to high salt induce hypertension and the aim of this study was to characterize the metabolic profiles of amino acids in SS rats. We evaluated whether these amino acids, especially L-phenylalanine, which is known to increase GCH activity and BHBN rats were purchased from Vital River (Beijing) and maintained with 12 h:12 h dark/light and free access to food and water. After adapting for 1 week, 7-week-old male rats were randomized into the low salt diet  and high salt diet  groups. Systolic blood pressure (SBP) was measured using tail-cuff methods with a computerized system  as previously described [The diet used in the present study was obtained from Jiangsu Xietong . Dahl salt-sensitive (SS) rats and SS.13escribed . All aniFor assessment of L-phenylalanine supplementation, all 7-week-old male SS rats were maintained on low salt diets and had free access to water. The L-Phe supplementation group was given free access to drinking water with 2.5% w/v L-Phe and low salt diets as described previously . After 4For free amino acids, 100 μl of separated serum was extracted with 200 μl of 8% sulfo-salicylic acid solution. After storage at -20°C for 30 min, the supernatants of the samples were collected after 10 min of centrifugation (13000 g at 4°C).The concentrations of the amino acids were determined using an automated amino acid analyzer . The analytical detector was set to measure a specific wavelength of 570 nm and 440 nm for proline and hydroxyproline. The buffer solutions, consisting of four different pH values , were purchased from Sykam (Germany). The flow rate of the amino acid analyzer was 0.6 ml/min, and the injection volume was 50 μl. The purchased standards of each individual amino acid were used for identification and quantification , and the concentrations of individual free amino acid values are expressed in μM. During the running of the samples, a separate delivery pump for the ninhydrin reagent automatically mixes these two solutions, which are kept under nitrogen in the amino acid analyzer. The flow rate for the ninhydrin solution was 0.30 mL/min (130°C).For determination of the effects of L-Phe supplementation on vascular reactivity, aortic rings were isolated with the integrity maintained from all groups at 37°C. Then, vessels were precontracted with phenylephrine. The response of the aortic vessels to ACh was determined by measuring diameter of aortic vessels, as previously described .4 levels were measured from the aortic vasculature and kidney using a commercial ELISA kit at 480 nm. Quantification of total nitrite was performed as previously reported using nitrate reductase at 550 nm. Superoxide levels were estimated by a lucigenin chemiluminescence-based assay as previously described [Tissues were homogenized in 5 volumes of HEPES buffer  and extracted by centrifugation . The supernatants were collected and measured by BCA protein assays (Thermo). The BHescribed . Tissue L-phenylalanine, tyrosine and dopamine in serum and the kidney were measured as previously described . Briefly5’-ATTTGTGGGAAGGGTCCA-3’, reverse: 5’-CAGATAACGCTGGCCTCA-3’) was obtained from BGI . The PCR conditions were as follows: 35 cycles of 94°C for 10 s, 60°C for 20 s, and 82°C for 15 s of fluorescence collection. Expression was quantified by the comparative CT method and normalized to GAPDH with three repetitions.Total RNA was extracted from the kidney, and cDNA was synthesized using TaKaRa kits following the manufacturer’s protocol. The expression of GCH1 was analyzed by RT-PCR with SYBR Green PCR Master Mix. Rat GCH1 . A two-way analysis of variance was utilized to determine the differences in parameters between SS and SS.13BN rats  than SS rats. Compared with low salt diets, high salt diets caused significant reduction of tyrosine in both SS rats and SS.13BN rats . By the end of the experiments, systolic blood pressure of the L-Phe supplementation group was 18 mmHg lower than that of the vehicle group. Besides, the urinary albumin was significantly decreased in HS-Phe group than HS group. Significant increase of serum nitrite was also detected in HS-Phe group compared with the hypertensive HS group  mRNA was no significant changes between LS group and HS group. Whereas, increased levels of GCH1 mRNA was observed in the L-Phe supplementation group compared with the high salt group . Althougalt diet .L-Phe is catalyzed to tyrosine via the enzyme of phenylalanine hydroxylase in vivo. The decreased level of serum L-phenylalanine in the serum of hypertensive rats was reversed in the HS-Phe group . AlthougBN rats, levels of glycine and methionine were increased in SS rats, which was consistent with previous reports in the plasma and kidney [In the present study, we quantitatively analyzed the amino acid contents and showed that only 8 of 27 amino acids responded to high salt in SS rats. Among these amino acids, branched-chain amino acids  are structural constituents of proteins that were significantly decreased in hypertensive rats. However, higher BCAA levels were associated with a higher risk of incident hypertension . Althougd kidney , 31. We 4 in the kidney and reduced NO production in the vasculature by uncoupling NOS. Confirming previous findings, we found that L-Phe supplementation reversed the inhibitory GCH1-BH4 complex in both the vasculature and kidneys, thus likely contributing to the increased NO production and decreased blood pressure [4, as an allosteric and essential cofactor for NO synthase, plays a critical role in the stabilization of the NO synthase dimer and increases the affinity of NO synthase for arginine [4, which led to increased sensitivity to high salt diets. Taken together, these data suggested that exogenous phenylalanine may increase BH4-dependent NO production, leading to decreased blood pressure in hypertensive SS rats.Elevated blood pressure has been demonstrated in SS rats with decreased NO . Althougpressure , 23. Furarginine , 34. The4 has been reported to be a powerful oxidant of O2• [4 with L-Phe supplementation [The production of superoxide by NO synthase plays an important role in the development of hypertension . BH4 hast of O2• . The obsentation . The eff4 or L-Arg [Endothelial function plays a key role in controlling blood pressure and the development of cardiovascular disease . Our resor L-Arg , 40. Thu4 levels [4 level in SS.13BN rats, which was reported previously [The deficiency in GCH activity was likely contributed to the reduced BH4 levels . Althoug4 levels . The unceviously , was conObviously, phenylalanine hydroxylase also could convert L-phe to tyrosine, which was further catalyzing the conversion of tyrosine to dopamine and adrenaline. Although levels of dopamine were not altered followed with L-Phe treatment, increased blood pressure were attenuated in HS-Phe group indicating that dopamine may be not involved in salt induced hypertension . Consist4 and nitrite, thus attenuating salt-induced hypertension in SS rats through the improved vascular and kidney function.We conclude that there were several altered amino acids in hypertensive SS rats that could contribute to salt-induced hypertension. L-Phenylalanine supplementation can directly affect levels of BH


Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria. Locked nucleic acids and 2′-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP–DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections. Antimicrobial resistance (AMR) is an increasing major public health concern, and the scientific community is struggling to find alternatives as we are entering the post-antibiotic era . During Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) such as locked nucleic acids (LNAs), able to resist the degradation by nucleases and with impressive binding affinity for complementary RNA, have the potential to modulate the expression of specific genes and hold promise as alternative drugs to conventional antibiotics ,6,7. TheLiposomes are currently one of the most biocompatible and widely used carriers in the pharmaceutical industry, with several liposomal formulations already on the market targeting human cells ,15,16. ITo treat septicemia, the liposomes not only need to deliver the ONs across the bacterial envelope, but also need to safely carry the ONs in human blood on their way to bacteria. The association of ONs with liposomes can be severely destabilized by human serum, due to the presence of lipases, nucleases and high-density proteins ,23. PolyH. pylori [Our group has pioneered the use of liposomes to deliver NAM-composed ONs into bacteria and already showed successful delivery by DOTAP–DOPE liposomes into . pylori . Being a. pylori ,28. In tN-(methoxy(polyethyleneglycol)-2000) (DSPE-PEG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (NBD-PE) were purchased from Avanti Polar Lipids . Chloroform, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride (Tris-HCl), sodium chloride (NaCl), Triton X-100 and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich . Paraformaldehyde was purchased from Fluka . Urea was acquired from VWR . Tryptic soy broth (TSB) was purchased from Merck Millipore .-trimethylammonium-chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-In order to study the efficiency of liposome delivery in different bacteria, a model NAM-ON that hybridizes with all eubacteria was used. As such, the ON EUB338 was chosen since it is complementary to a conserved 16S rRNA sequence present in the domain Bacteria. The ON sequence used is composed of the NAMs locked nucleic acids (LNA “+”) and 2′-OMethylRNA (2′OMe “m”) and possesses phosphorothioate (PS) internucleotide linkages (“*”): 5′+T*mG*mC*+C*mU*mC*+C*mC*mG*+T*mA*mG*+G*mA-3′. The ON was fluorescently labeled at 5′ with HiLyte 488 and was purchased from Eurogentec .DOTAP and DOPE liposomes were prepared  as reporv/v) of total lipids was added in the initial phospholipid mixture. For the fusion assays, additionally to Rh-PE, NBD-PE was also added to the initial phospholipid mixture so that the final concentration of the labeled lipids resulted in 0.5% (v/v) each. In both cases, the liposomes were diluted to 1 mM (each lipid) in HEPES.For interaction and fusion studies of the liposomes with different bacterial envelopes, empty liposomes were used. For the initial interaction assay based on epifluorescence microscopy, Rh-PE at a final concentration of 0.5% . The complexation was achieved after incubation for 30 min, at room temperature.Post-insertion of PEG lipids (2 kDa DSPE-PEG) was performed to obtain post-PEGylated liposomes or lipoplexes . Brieflyg, and the resulting serum was portioned into 500 μL aliquots and stored at −20 °C until use.Human serum collected from a healthy donor was obtained from the Ghent University Hospital and added to Venosafe 6 mL tubes containing gel and clotting activator . The tubes were centrifuged for 10 min at 4000× To understand if the formulation is adequate for intravenous administration, the stability of complexation between the liposomes and ONs was characterized by single-color FCS upon incubation in undiluted human serum. T is the percentage of molecules in triplet state and FCS is a microscopy-based technique that monitors the fluorescence fluctuations of the diffusing molecules in a focal volume of a confocal microscope. The degree of mobility of the diffusing species and their size is determined by the duration of the fluctuations. From the fluorescence fluctuations, an autocorrelation curve on curve ,30. The on curve ,32 were To investigate liposomes’ interaction with different bacterial envelopes (Gram-negative and Gram-positive), labeled liposomes were incubated with bacteria and visualized with an epifluorescence microscope. Rh-PE-labeled PEGylated and non-PEGylated DOTAP–DOPE liposomes were prepared using the methodology described above.Escherichia coli K12 MG1655, Staphylococcus aureus Mu50 ATCC 700699, Acinetobacter baumannii NCTC 13423, Enterococcus faecium CECT 410, Klebsiella pneumoniae ATCC 11296 and Pseudomonas aeruginosa PAO1 were selected due to their relevance in blood infections and multiresistance. Bacteria were grown overnight in TSB broth at 37 °C and diluted to an OD600 of 0.680 in HEPES buffer (pH 7.4). The bacterial suspension was mixed with Rh-PE liposomes (with or without PEG), resulting in a concentration of 50 µM of the total lipids in the mixture and a bacterial concentration (OD600) of 0.6. After 1 h incubation with shaking (180 rpm) at 37 °C, the suspension was diluted in filtered distilled water to a final OD600 of 0.1. Smears were prepared on microscope glass slides (20 µL per slide well) by drying at 37 °C. The bacteria were observed on a Nikon Eclipse Ti SR epifluorescence microscope , equipped with a QImaging Retiga R1 monochromatic camera  and a Nikon Plan-Apo 100 × 1.45 N.A. oil immersion objective lens . Rh fluorescence was visualized using a G-2A longpass filter  and maintaining the exposure time and the excitation intensity among the different samples; ten pictures of each sample were taken randomly, covering all the areas of the sample. The images were processed with NIS-Elements Advanced Research .To further understand the extent of interaction between liposomes and the different bacterial envelopes, a lipid-mixing assay based on fluorescence resonance energy transfer (FRET) was performed to quantify fusion .E. coli, S. aureus, A. baumannii, E. faecium, K. pneumoniae and P. aeruginosa were grown to their middle exponential phase, followed by a resuspension in previously heated (37 °C) 20 mM HEPES buffer (pH 7.4), resulting in a final OD600 of 0.680. The bacterial suspension was incubated at 37 °C with Rh-PE/NBD-PE liposomes (with or without PEG) at a final total lipid concentration of 50 µM and bacterial OD600 of 0.6. Every 10 min, 100 µL of each mixture was mixed in 100 µL of HEPES buffer and distributed into a 96-well plate. The fluorescence intensity of Rh was measured in a FLUOstar Omega Microplate Reader  at 590 nm under a steady-state excitation of 470 nm. After 60 min incubation, Triton X-100 detergent (0.2% (v/v) was added to the mixture, in order to completely disrupt the liposomes and lead to a maximal lipid probe dilution, and the final rhodamine fluorescence intensity was measured.Briefly, tF is the fluorescence intensity of Rh at each time point and F0 and maxF are its initial and final fluorescence intensity, respectively. The final fluorescence intensity was achieved after the addition of Triton X-100 detergent.The obtained percentage of fusion was calculated using the following Equation (3) :(3)% FusThe ability of the PEGylated liposomes to internalize NAM-ONs in Gram-positive and Gram-negative bacteria was studied using fluorescence in situ hybridization (FISH) followed by bacterial membrane staining.E. coli K12 and S. aureus Mu50 were grown overnight in TSB broth at 37 °C followed by a dilution to nearly 1.6 × 106 cells/mL and grown until the mid-exponential phase at 37 °C. FISH was then applied. Traditional in vitro FISH starts with a permeabilization/fixation step, needed to render the bacterial envelope permeable to the ONs, based on pretreating the bacteria with chemicals that are toxic for in vivo application [6 cells/mL was centrifuged for 15 min at 8600× g and resuspended in HEPES buffer containing PEGylated lipoplexes . The mixture was incubated for 1 h, at 37 °C, and centrifuged for 10 min at 8600× g. The bacterial cells were washed in 500 μL of washing solution  Triton-X, pH 10) for 15 min, at 37 °C, and centrifuged again . The bacteria were resuspended in 1% (v/v) DiD membrane dye in ethanol and incubated for 5 min at 37 °C. After centrifugation , bacterial cells were resuspended in sterile distilled water, and 20 μL of each sample was placed on a glass slide well and dried at 37 °C for microscopy visualization.lication ,36,37. Ilication . The bacMicroscope images were taken using a C1si laser scanning confocal microscope (CLSM)  and a 100x oil immersion objective . HiLyte 488 and DiD were excited by a 488 and 640 nm diode laser . At least 10 images were acquired with the NIS-Elements AR software  using an EMCCD camera .E. coli and S. aureus, after their contact with the lipoplexes, the bacterial cells were fractioned into a membrane fraction and a cytosol fraction, and the HiLyte 488 fluorescence intensity was measured in a FLUOstar Omega Microplate Reader . Overnight inocula of E. coli K12 and S. aureus Mu50 were diluted in HEPES buffer to an OD600 of 0.1, in the presence of PEGylated lipoplexes for 1 h, at 37 °C. The amount of lipoplexes added was such that 400 nM of labeled ONs were present in the final bacteria–lipoplexes mixture. Thereafter, a fractionation protocol adapted from Bandula et al. was followed [g, 20 min), resuspended in 10 mM Tris–150 mM NaCl (pH 7.4) and washed with 50 mM Tris (pH 7.6) to remove lipoplexes that did not interact with bacteria. To obtain the fraction associated with the outer membrane (membrane fraction), bacteria were centrifuged  and resuspended in 50 mM Tris buffer solution containing 0.05% (v/v) Triton X-100 (pH 7.6), for 1 h, at room temperature (RT). After a new centrifugation (same conditions), the fluorescence intensity of the supernatant (membrane fraction) was measured with the fluorometer. To obtain the fraction associated with the cytosol, bacteria were centrifuged, and the pellet was resuspended in a more astringent 50 mM Tris buffer containing 1% (v/v) Triton X-100 (pH 7.6), for 1 h, at RT. After the last centrifugation , the fluorescence of the supernatant (cytosol fraction) was measured. The percentage (%) of ONs in each fraction was calculated using the following Equation (4):To confirm the cellular localization of the labeled NAM-ONs in followed . In brieAs a control, the same fractionation protocol was applied to bacteria stained only with DAPI (a cell-permeable dye known to stain the cytosol by binding to DNA).p-value of ≤ 0.05 was considered as significant .Experiments were performed in triplicates on independent days. Significance between the means of the experimental groups was evaluated using two-way analysis of variance (ANOVA), applying Sidak’s multiple-comparison test. The software GraphPad Prism 7 was used and a Cationic liposomes, constituted of DOTAP and DOPE in a 1:1 mol ratio, were prepared following the lipid hydration method ,39. AsseRh/NBD-labeled liposomes, prepared for the interaction studies, had an identical average zeta potential as their unlabeled counterparts and a hydrodynamic diameter of 86–106 nm. The final zeta potential of PEGylated labeled liposomes was around +16–18 mV and the average hydrodynamic diameter was 93–113 nm.Oftentimes, the stability of formulations in biologically relevant fluids is disregarded in biochemical and microbiology studies, causing premature failure of novel antimicrobial strategies. In order to treat bloodstream infections, commonly caused by multidrug-resistant bacteria, human serum needs to be considered as the first delivery barrier. Upon intravenous administration, lipoplexes will come into contact with blood, and interactions with serum components may result in premature release of the NAM-ONs from the lipoplexes . As thisv/v) DSPE-PEG, more than 90% release of siRNA was reported after 1 h incubation in human serum by Dakwar et al. [r et al. . Howeverr et al. , is a prEscherichia coli K12, Staphylococcus aureus Mu50, Acinetobacter baumannii, Enterococcus faecium, Klebsiella pneumoniae and Pseudomonas aeruginosa, and incubated with empty liposomes, with and without PEG. The interaction between bacteria and empty liposomes was analyzed using epifluorescence microscopy  and the bacterial membranes, using a FRET-based lipid mixing assay ,47,48. TS. aureus and found that the fusogenic liposomes were the most successful in inhibiting bacterial growth. They also found that free vancomycin had a better inhibitory effect in the early stages of biofilm formation than the liposomal formulation, but the reverse happened in a mature biofilm, once the peptidoglycan layers became thicker. They postulated that the liposomes had an increased ability to penetrate the peptidoglycan layers, whereas the free vancomycin remained trapped in the cell wall [From ell wall . Concernell wall .E. coli and S. aureus . While PEGylation of lipoplexes improves their stability in biofluids, it has, however, been reported that PEGylation also lowers the interaction of liposomes with cells by limiting the electrostatic and fusogenic interaction of the cationic liposomes with the negatively charged membranes [E. coli and the Gram-positive S. aureus were the bacteria with lower percentages of fusion with PEGylated liposomes, which is also denoted by a lower rhodamine fluorescence intensity in The results presented in embranes ,54,55. Nembranes . Interesp > 0.001). The only exception is related to the fusion of PEGylated liposomes with S. aureus and E. coli, which was very residual (p > 0.05). These results indicate that the events of self-fusion do not significantly promote the decrease in FRET that was observed when fusion of liposomes with bacteria was investigated. Additionally, fluorescence did not vary significantly over time when rhodamine emission was monitored under rhodamine excitation (530 nm), instead of NBD (470 nm), indicating that the decrease in FRET was not promoted by photobleaching (not shown).A control using liposomes was performed to evaluate the extent of self-fusion events. Liposomes, with and without PEG, were tested with both fluorophores against their unlabeled counterparts . The resE. coli and S. aureus to test if the significantly lower interaction with post-PEGylated liposomes negatively affects intracellular delivery of NAM-ONs, as PEG appears to be pivotal in future in vivo applications. Using E. coli and S. aureus as model Gram-negative and Gram-positive bacteria, respectively, we investigated (i) the intracellular delivery of NAM-ONs using confocal microscopy and (ii) their relative distribution within the cytosol and membrane by bacterial cell fractionation.We aimed to investigate how much of the observed interaction and fusion is needed to successfully deliver NAM-ONs into bacteria. Therefore, we used E. coli and S. aureus. This observation proves that the post-PEGylated lipoplexes are at least partially able to release ONs in both Gram-negative and Gram-positive bacteria, mediated by liposomal fusion with the envelope.Visualization of the ONs internalization was obtained after performing FISH with the post-PEGylated lipoplexes (carrying HiLyte 488 ONs) followed by membrane staining with DiD. As presented in E. coli and S. aureus, respectively, with the remaining amount colocalized with the membrane. As expected, the DAPI control was mostly found in the cytosolic fraction (68–78%), validating the obtained fractionation results. The small DAPI fraction associated with the membrane is in agreement with previous reports [To quantify the relative amount of ONs delivered in the bacterial cytosol, we performed bacterial cell fractionation after contact with the post-PEGylated lipoplexes B. We fou reports .E. coli and S. aureus (Overall, the results show that fusion of lipoplexes does not necessarily lead to the delivery of all the transported cargo. Moreover, despite the apparent low levels of labeled ONs in the cytosol of . aureus B, post-P. aureus A.Whether such amount of delivered NAM-ONs is sufficient for future successful antisense therapy is unknown, as there are no studies showing the amount of internalized ASOs that is needed to efficiently inhibit bacterial gene translation. Nonetheless, the fact that the number of copies of rRNA in a bacterium is significantly higher than mRNA is promising .After decades of study, the therapeutic potential of ONs remains largely unrevealed. Attempts to fundamentally explain the biological activity and the biophysical properties of nucleic acid loaded carriers are scarce but greatly needed to allow the design of better delivery systems for nucleic acids. DOTAP–DOPE liposomes have been previously shown to efficiently deliver ONs into eukaryotic cells ,59,60 anThe results presented in this study are promising as they indicate that post-PEGylated DOTAP–DOPE liposomes successfully fuse with both Gram-negative and Gram-positive bacteria. Additionally, the formulation may be considered to treat bloodstream infections as it remains stable in human serum. Therefore, ONs that are designed to hybridize with essential genes or genes associated with antibiotic resistance can potentially be delivered into different clinically relevant bacteria. Future research will focus on dose-dependent assays using ONs to evaluate their antimicrobial efficiency and test their in vivo performance.


Neisseria gonorrhoeae (N. gonorrhoeae) is now recognized as a commonly reported sexually transmitted pathogen, and the increasing drug resistance of N. gonorrhoeae has become a serious public health problem. The accuracy of molecular detection for detecting moderate-level azithromycin resistance is not well-established. We summarized the data from studies of the N. gonorrhoeae 23S rRNA mutation at position 2611 with azithromycin resistance to determine the relationship between the mutation and resistance. Methods and Findings: In this systematic review and meta-analysis, two researchers independently searched six databases for studies with data for the azithromycin minimum inhibitory concentrations (MICs) and the 23S rRNA mutation C2611T of each N. gonorrhoeae isolate. Since the breakpoint of moderate-level resistance to azithromycin (ML-AzmR) was not determined, we divided the moderate level into two groups according to the range of MICs (moderate resistance limited to 2–128 mg/L or 4–128 mg/L) for data extraction. A random-effects model was used to calculate the pooled sensitivity rate, the specificity rate, the pooled positive likelihood ratio (PLR), the negative likelihood ratio (NLR), and the diagnostic odds ratio (DOR). Meta-regression analyses by detection method, isolates sampling (a random sample or not), location, and sample size were performed to explore the possible causes of heterogeneity. The potential publication bias of the included studies was conducted by the Deeks’ test. We included 20 studies in our study: 20 studies have data of N. gonorrhoeae with MICs between 2 and 128 mg/L with mutation or without mutation at position 2611(4759 samples), and 14 studies have data of N. gonorrhoeae with MICs between 4 and 128 mg/L (3367 samples). In the group with the moderate level of 2–128 mg/L, the pooled sensitivity rate of the molecular assays was determined to be 71.9% , the pooled specificity rate was 98.7% , and the DOR ranged from 55.0 to 351.3 . In the 4–128 mg/L group, the pooled sensitivity rate was 91.9% , the pooled specificity rate was 95.9% , and the DOR ranged from 41.9 to 364.1 . Conclusion: Through this meta-analysis, we found that the C2611T mutation of 23S rRNA is valuable for the molecular diagnostic of moderate-level azithromycin resistance (ML-AzmR) in N. gonorrhoeae, especially when the moderate level is set at 4–128 mg/L. This rapid molecular detection method can be used for the rapid identification of ML-AzmR isolates in the clinic.Background:  Neisseria gonorrhoeae (gonococcus) is the etiologic agent of gonorrhea, a sexually transmitted infection (STI) that remains a major global public health concern [ concern . In Chin concern . The glo concern .N. gonorrhoeae has been identified as an emerging public health problem. In 1990, the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP) was established to monitor gonococcal antimicrobial resistance (AMR) worldwide [N. gonorrhoeae strains with resistance to azithromycin [N. gonorrhoeae isolates can be categorized into high-level azithromycin-resistant (HL-AzmR), moderate-level azithromycin-resistant (ML-AzmR), and low-level azithromycin-resistant (LL-AzmR). The azithromycin MICs of HL-AzmR N. gonorrhoeae isolates are commonly defined as ≥256 mg/L. However, it is still controversial whether the lower limit of ML-AzmR N. gonorrhoeae isolates’ MICs is 2 or 4 mg/L [N. gonorrhoeae (PPNG), tetracycline-resistant N. gonorrhoeae (TRNG), and chromosomally mediated penicillin-resistant N. gonorrhoeae (CMRNG) [N. gonorrhoeae.In recent years, gonococcus has become progressively resistant to a wide range of antibiotics, include tetracyclines, penicillin, and sulfonamides . As a reorldwide . Nowadayorldwide  and the orldwide . Howeverhromycin . On the  (CMRNG) , but notN. gonorrhoeae is primarily associated with 23S rRNA point mutations. The 23S rRNA of N. gonorrhoeae is an important target for azithromycin to bind to and exert its toxicological effect by interfering with mRNA. There are four alleles in the operon gene of the V region of 23S rRNA. The mutation sites are C2611T and A2059G (E. coli coding system), but C2611T is more common. It is generally thought that the mutation of the 2611 site is related to the moderate level of azithromycin resistance, while the A2059G mutation can lead to high levels of azithromycin resistance [The resistance to azithromycin of N. gonorrhoeae. Through this study, we provide a basis for further research into the rapid and accurate detection of 23S rRNA point mutant N. gonorrhoeae using the molecular detection method.So far, there is no clear study on the accuracy of 23S rRNA mutation C2611T detection for azithromycin resistance. In this study, we systematically evaluated the sensitivity and specificity of molecular detection targeting the mutation C2611T for detecting middle-level azithromycin resistance in This study was registered with PROSPERO, number CRD42021248296.The present study was carried out following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines . Two resN. gonorrhoeae isolates; (3) the study indicated the results of molecular assays targeting position 2611 of the 23S rRNA gene.Search results were first screened based on the title and abstract, and any studies that appeared to meet the eligibility criteria, or where eligibility was unclear, progressed to full-text screening. Studies included in our meta-analysis had to be consistent with the following criteria: (1) the research was published in English or Chinese; (2) the study indicated the number of moderate-level azithromycin resistance and non-moderate-level azithromycin resistance N. gonorrhoeae isolates’ MIC is 2 or 4 mg/L, so we divided them into two groups (the moderate-level drug resistance breakpoint was 2–128 or 4–128 mg/L) to extract the article data for meta-analysis. Information extracted from all studies included: (1) title, first author, publication year, study country, study period; (2) the technique used for detecting the mutation C2611T; (3) isolates selection (was the sampling continuous or random); (4) the numbers of true positives (TPs), false positives (FPs), false negatives (FNs), and true negatives (TNs)  . Some ofTo assess the methodological quality of the eligible studies, the QUADAS2 tool was used to examine bias in studies of diagnostic accuracy by two independent investigators (Z.Q. and L.J.) . AccordiN. gonorrhoeae detected by the agar dilution method and the E-test method as the gold standard. The numbers of ML-AzmR N. gonorrhoeae isolates with mutation or without mutation, and non-ML-AzmR isolates with mutation or without mutation were defined as true positive (TP), false negative (FN), false positive (FP), and true negative (TN), respectively. Meta-Disc 1.4 [Neisseria gonorrhoeae isolates. The heterogeneity was evaluated by performing the Q test and calculating I2 values. I2 > 50%, or a p-value for heterogeneity less than 0.1, indicated high heterogeneity. To find the source of the heterogeneity of the results, the Spearman correlation coefficient and meta-regression analysis were applied. Meta-regression analysis was performed according to the detection method, isolates sampling (was a random sample or not), location, and sample size. The Deeks’ funnel plot asymmetry test was used to detect potential publication bias.We took the moderate-level azithromycin resistance phenotype of Disc 1.4  and StatA total of 422 potentially relevant abstracts were identified, of which 259 were duplicates and thus removed. The remaining 259 abstracts were assessed; 197 of them were irrelevant articles, reviews, and case reports and were subsequently excluded. The remaining 62 full-text articles were assessed for eligibility and 42 of them were excluded because they consisted of duplicated data published in a different language, or they did not indicate azithromycin MICs or the 23S rRNA mutant at position 2611. There were 20 studies that met the inclusion criteria for the meta-analysis ,32,33,34Risk of bias results are summarized in N. gonorrhoeae isolates (MICs was 2–128 mg/L) based on the mutation C2611T. The sensitivity rates in these studies ranged from 34.5% to 100.0%. The pooled sensitivity rate of the molecular assays was determined to be 71.9%  (Twenty studies presented data for determining the sensitivity rate of detection of ML-AzmR 7.6–74%) A, and th7.6–74%) A. Pooled7.6–74%) , whereas7.6–74%) . The DOR7.6–74%) . An sROC7.6–74%) A.N. gonorrhoeae isolates (MICs was 4–128 mg/L) based on the mutation C2611T. The sensitivity rates in these studies ranged from 40.0% to 100.0%. The pooled sensitivity rate of the molecular assays was determined to be 91.9%  (Fourteen studies showed data for determining the accuracy of the detection of ML-AzmR 9–94.2%) B, and th9–94.2%) B. Pooled9–94.2%) , whereas9–94.2%) . The DOR9–94.2%) . An sROC9–94.2%) B.p = 0.08) indicated that there was no threshold effect. Further meta-regression analysis based on continuous variables, such as the detection method, isolates sampling (a random sample or not), location, and sample size indicated, that these factors were not the sources of heterogeneity  indicated that there was no threshold effect. Further meta-regression analysis based on continuous variables such as the detection method, isolates sampling (as a random sample or not), location, and sample size indicated that these factors were not the sources of heterogeneity  to extract the article data. In the 2–128 mg/L group, the meta-analysis of the mutation of C2611T for the diagnosis of middle-level azithromycin resistance showed that the pooled sensitivity was 71.9%, the pooled specificity was 98.7%, and the missed diagnosis rate was 28.1%. In the 4–128 mg/L group, the pooled sensitivity was 91.9%, the pooled specificity was 95.9%, and the missed diagnosis rate was lower than that of the 2–128 mg/L group. We also combined sensitivity and specificity rates to create the sROC curve. The AUC of the 2–128 mg/L group was 0.93, and the AUC of the 4–128 mg/L group was 0.96, indicating that the accuracy of the mutation of C2611T for the diagnosis of middle-level azithromycin resistance was 96% in the group of 4–128 mg/L, which was higher than that of the 2–128 mg/L group. On the basis of the above data, molecular detection of the C2611T mutation has high accuracy in the diagnosis of ML-AzmR N. gonorrhoeae, especially when the MIC range is 4–128 mg/L.A total of twenty studies were included in the meta-analysis. The premise that the lower limit of the ML-AzmR Neisseria gonorrhoeae. Previous studies [N. gonorrhoeae by combining two molecular detection methods. Thus, with the development of molecular detection kits based on these two mutations, the 23S rRNA point mutation of azithromycin-resistant N. gonorrhoeae will become a clinically and routinely detected resistance phenotype such as PPNG and TRNG. In terms of clinical significance, the highly accurate molecular detection of clinical isolates, low-level azithromycin-resistant, and moderate- and high-level azithromycin-resistant bacteria can be identified in the early clinical stage, improving efficiency and precision in the treatment of patients. For low-level, drug-resistant, and azithromycin-sensitive strains, a single 2 g dose of azithromycin is effective. For moderate- and high-level drug-resistant strains, other drugs, such as third-generation cephalosporins, need to be used [This study has many advantages. Methodologically, this is the first analysis to explore the relationship between the C2611T mutation and moderate-level azithromycin resistance. Our results confirm that the C2611T mutation is an important factor leading to the moderate level of azithromycin resistance of  studies  proved t be used ,37,38.There are limitations to this study. The meta-analysis shows that there is a high degree of heterogeneity among the included studies. The Spearman correlation analysis suggests that there is no threshold effect. Meta-regression analysis shows that the detection method, isolates sampling, location, and sample size are not the sources of heterogeneity. A possible explanation for the heterogeneity is that the sample size of some studies was less than 100. However, the sample size after meta-regression analysis was not the source of heterogeneity. More high-quality studies with larger sample sizes may be needed in the future.N. gonorrhoeae antibiotic resistance. This meta-analysis shows that the molecular diagnostic accuracy of the 2611 mutation of 23S rRNA is high, especially from 4–128 mg/L. Molecular detection methods have promising potential for use as a diagnostic kit for the rapid identification of ML-AzmR isolates in the clinic. More high-quality studies with larger samples are needed to confirm this finding.Molecular detection methods can quickly detect the specific gene mutation of clinical isolates. Through the efficient detection of resistant gene mutations, patients can be given the appropriate concentration of antibiotics, which can effectively inhibit the emergence of


XDH (type I) or MOCOS (type II) genes. Thirteen Israeli kindred (five Jewish and eight Arab) and two isolated cases from Germany were studied between the years 1997 and 2013. Four and a branch of a fifth of these families were previously described. Here, we reported the demographic, clinical, molecular and biochemical characterizations of the remaining cases. Seven out of 20 affected individuals (35%) presented with xanthinuria-related symptoms of varied severity. Among the 10 distinct variants identified, six were novel: c.449G>T (p.(Cys150Phe)), c.1434G>A (p.(Trp478*)), c.1871C>G (p.(Ser624*)) and c.913del (p.(Leu305fs*1)) in the XDH gene and c.1046C>T (p.(Thr349Ileu)) and c.1771C>T (p.(Pro591Ser)) in the MOCOS gene. Heterologous protein expression studies revealed that the p.Cys150Phe variant within the Fe/S-I cluster-binding site impairs XDH biogenesis, the p.Thr349Ileu variant in the NifS-like domain of MOCOS affects protein stability and cysteine desulfurase activity, while the p.Pro591Ser and a previously described p.Arg776Cys variant in the C-terminal domain affect Molybdenum cofactor binding. Based on the results of haplotype analyses and historical genealogy findings, the potential dispersion of the identified variants is discussed. As far as we are aware, this is the largest cohort of xanthinuria cases described so far, substantially expanding the repertoire of pathogenic variants, characterizing structurally and functionally essential amino acid residues in the XDH and MOCOS proteins and addressing the population genetic aspects of classical xanthinuria.Classical xanthinuria is a rare autosomal recessive metabolic disorder caused by variants in the  Classical xanthinuria is a rare, autosomal recessive metabolic disorder characterized by impaired xanthine dehydrogenase XDH; EC 1.17.1.4) activity that results in a lack of uric acid (UA) formation and accumulation of its precursors, xanthine (X) and hypoxanthine (HX) .17.1.4 a. ClassicXDH gene [MOCOS, alias HMCS) cause combined XDH and Aldehyde Oxidase  deficiency [XDH gene is located on chromosome 2p23.1 (reference [MOCOS gene is located on chromosome 18q12.2 (NC_000018.9) and encodes an mRNA of 2.7 kb (NM_017947.1) and a protein of 888 amino acid residues (NP_060417.4). The MOCOS enzyme (EC 2.8.1.9) catalyzes the conversion of the oxo-form to the sulfido-form of Moco that is essential for the activity of both XDH and AO [rcsb.org).Two genetically distinct types of classical xanthinuria can be discerned. Type I xanthinuria MIM 278300) is caused by variants in the 00 is cauficiency . The 80.eference , NC_0000eference ,7. The aeference ,10. The b NM_01797.1 and aH and AO . To dateIn the past, we described the molecular basis of classical xanthinuria in five families ,13,14. HNine families (designated F1–F9 in order of their presentation) and two sporadic cases (G1 and G2) referred to our laboratory between the years 1997 and 2013 and 34 individuals of Yemenite-Jewish extractions consecutively admitted to the Rheumatology Department at the Tel Aviv Medical Center from April 1997 to January 1998 were studied.Testing for the levels of UA by routine clinical biochemistry analysis and analysis of X and HX, as well as the allopurinol loading test  by high-www.thermofisher.com accessed on 4 April 2021) was incorporated into the PCR reaction mix at a final concentration of 1.5 µM.DNA analysis was performed on 76 samples belonging to the affected families/cases and 34 control samples of Yemenite-Jewish origin. DNA was manually extracted from peripheral blood . Analysiwww.ncbi.nlm.nih.gov accessed on 6 June 2020) was queried for the putative bovine MOCOS cDNA (GeneBank ABO36422) [www.ncbi.nlm.nih.gov accessed on 6 June 2020) was then queried with this cDNA sequence, and five high-score genomic clones containing several unordered pieces that mapped to chromosome 18q12 were retrieved: DDBJ: AP001321.1, AP001159.1, AP001158.1, AC023043.2 and AP001155.1. The order of the exons was obtained by alignment with the putative bovine MOCOS gene [At the start of this project, the human gene responsible for type II xanthinuria was still unknown; therefore, the following strategy was used for variant detection in the suspected type II xanthinuria patients. The human cDNA database Entrez-Protein (BO36422)  using thBO36422) . This seCOS gene . PrimersWhole-exome DNA sequencing for the detection of variants in families F4 and F9 was performed by Macrogen . Exonic and adjacent intronic regions were enriched from genomic DNA derived from peripheral blood via Agilent SureSelect v5, followed by next-generation sequencing (NGS) on the Illumina HiSeq 4000. Variants were detected, analyzed and reported using the Variantyx Genomic Intelligence platform.http://varnomen.hgvs.org/bg-material/refseq/ accessed on 4 April 2021), numbering the A of the initiation methionine as +1 in the respective nucleotide reference sequences (NM_) and the corresponding amino acid as +1 in the respective protein reference sequences (NP_).Nomenclature: all variants were described according to the Human Genome Variation Society (HGVS) guidelines (www.ncbi.nlm.nih.gov>clinvar accessed on 10 May 2021). The pathogenicity of the missense variants was evaluated using the Protein Variation Effect Analyzer Software Tool  that provides PROVEAN and SIFT predictions. When the PROVEAN score was equal to or below a predefined threshold (−2.5), the protein variant was predicted to have a “deleterious” effect. If the PROVEAN score was above the threshold, the variant was predicted to have a “neutral” effect. SIFT scores ranged from 0 to 1. The amino acid substitution was predicted as “damaging” if the score was under 0.05 and “tolerated” if the score was over 0.05. In addition, the PolyPhen-2 in silico prediction tool (http://genetics.bwh.harvard.edu/pph2/index.shtml accessed on 10 May 2021) was used. The PolyPhen-2 score ranged from 0.0 (tolerated) to 1.0 (deleterious). Variants with scores ranging 0.0–0.15 were predicted to be benign, variants with scores ranging 0.15–1.0 were possibly damaging and variants with scores ranging 0.85–1.0 were probably damaging.The novelty of the identified variants was verified in the ClinVar database (consurf.tau.ac.il/) bioinformatics tool [The degree of conservation and solvent accessibility of the amino acid residues in the MOCOS protein was evaluated as formerly described . The Conics tool  was usedA. thaliana AtXDH1 expression constructs, expression and purification of AtXDH1 proteins, as well as protein analysis, were described previously by Zarepour et al. [The generation of r et al. . Primers6 tag for affinity purification. The expression construct for the MOCOS-NifS/Thr349Ile variant was generated accordingly, albeit with a mutated open reading frame generated via PCR mutagenesis using the primers: MOCOS-NifS/T349I_for (5′-GAG AAT ATA AAG CAG CAC ATC TTC ACC TTG GCT CAG-3′) and MOCOS-NifS/T349I_rev (5′-CTG AGC CAA GGT GAA GAT GTG CTG CTT TAT ATT CTC-3′).For the expression of wild-type MOCOS-NifS, the first 1524 bp of the full-length human MOCOS open reading frame were cloned into the pQE80 plasmid  according to standard cloning procedures to allow for the heterologous production of a 508-aa MOCOS-NifS protein with an N-terminal fused HisEscherichia coli BL21 (de3) cells. Cells were grown aerobically in LB medium in the presence of 50-µg/mL ampicillin at 37 °C to an A600 = 0.5 before induction. Protein expression was induced with 25-µM isopropyl-β-D-thiogalactopyranoside (IPTG) after the addition of the PLP precursor pyridoxin to the medium  to support the synthesis of the PLP cofactor that is required for MOCOS-NifS functionality. After culturing for 20 h at 26 °C, cells were harvested by centrifugation  and stored at −70 °C until use. Prior to cell lysis, cells were resuspended and thawed in 5 volumes of lysis buffer . Cell lysis was achieved by passaging through a French pressure cell followed by sonication for 5 min on ice. After centrifugation at 50,000× g for 30 min at 4 °C, His6-tagged proteins were purified on a nickel-nitrilotriacetic acid superflow matrix  under native conditions at 4 °C in buffers of pH 9.3, according to the manufacturer’s instructions, and eluted in elution buffer . After purification by affinity chromatography, the recombinant NifS-like domain of MOCOS was subjected to PLP quantification and a cysteine desulfurase activity analysis.The standard expression of MOCOS-NifS and its Thr349Ile variant was performed from the pQE80 plasmid  in freshly transformed A. thaliana [To determine the content of protein-bound PLP, PLP-specific absorption at 420 nm was measured in preparations of MOCOS-NifS and its Thr349Ile variant and compared to a PLP standard curve (0–350-µM PLP) as described earlier for the MOCOS-NifS homolog ABA3-NifS from thaliana .The determination of the cysteine desulfurase activity was performed basically as described previously by Lehrke et al. , with thCloning, expression and purification of the MOCOS-CT proteins was described earlier by Giles et al. , while qThe genealogical history of the affected families was studied by the method of “networking”  using seNine Israeli families with 18 affected individuals and two sporadic cases referred from Germany are described. Pedigrees of the studied families, including ethnic origins, the gene in which disease-causing variants were detected and nonrelated congenital disorders, are depicted in XDH gene and three (two novel) variants in the MOCOS gene causing type I and type II xanthinuria, respectively, along with the variants previously reported by us are shown in The clinical features of the affected individuals are summarized in XDH gene showed that the affected individuals were heterozygous for the tested markers (XDH gene could not be excluded (not shown).The parents and the oldest sibling in family F1 were born in the city of Ta’izz (Shar’ab District in the Southwest of Yemen). The index case was diagnosed with severe urolithiasis at the age of 40 years and presented with joint pain at the age of 41. This clinical combination of joint pain and urolithiasis raised the diagnostic possibility of gout. However, routine biochemical testing revealed undetectable levels of UA in the serum and urine. Classical xanthinuria was confirmed by high levels of urinary xanthine (X) and hypoxanthine (HX), and three additional affected family members were identified by similar biochemical findings . There w markers , suggestFamily F3 was apparently unrelated to family F1, yet originated from the same city, Ta’izz, in Yemen. The proposita, a 69-year-old asymptomatic female, presented with obesity and non-insulin-dependent diabetes mellitus (NIDDM) and was incidentally found to have undetectable levels of UA in the serum and urine. There was no known history of renal stones in this family. The index case reported that her parents were second- or third-grade cousins. Xanthinuria was confirmed by high levels of X and HX in the urine , and typFamily F8 was a third, apparently unrelated, Yemenite-Jewish family, originating from the same area as families F1 and F3. The propositus, a 76-year-old male, was followed for benign prostate hypertrophy, type 2 diabetes mellitus, adenocarcinoma of the colon and metastatic transitional cell carcinoma (TCC) of the bladder, from which he died at the age of 85. There was no evidence for urolithiasis, though near his death, particulate material was noted in the ureters in an ultrasound report. Xanthinuria was suspected because of very low levels of UA in the blood and urine . The parXDH gene were nonconclusive, screening for variants by a SSCP analysis in both the XDH gene and a putative MOCOS gene (see Methods) was initiated. When type II xanthinuria was diagnosed by an allopurinol loading test in family F3, we assumed that these families may share a common variant in the MOCOS gene. Indeed, the SSCP analysis and direct sequencing resulted in the identification of a c.1046C>T (p.(Thr349Ileu)) transition in exon 6 of the putative MOCOS gene in both families , a six-month-old male, presented with renal calculi and splenomegaly of unknown etiology. He was found to have undetectable levels of UA in the serum and strongly decreased levels of UA together with elevated levels of X and HX in the urine . Low UA  patient . A high-An unusual result was noted in the segregation analysis in family F2. Using 14 SSRs covering 10.01 cM revealed that the affected siblings (V-2 and V-3) had identical genotypes, yet the run of homozygosity (ROH) of 2.86 cM  was unexXDH gene  was also homozygous for this genetic variant. The variant was confirmed by Sanger sequencing (not shown).Recently, trio-exome sequencing was performed in this family to search for a diagnosis of his affected brother (V-6), who presented with severe global developmental delay. This analysis revealed that V-6 and his father were homozygous for the genetic variant NM_000379.3:c.913del (p.(Leu305fs*1)) in the Family F5 was a large Bedouin-Arab family living in a town located in Southern Israel. The index case, a 4-year-old boy, presented with kidney stones and undetectable levels of UA in the serum and urine . The UA Family F7 was also of Bedouin-Arab origin from Southern Israel. Families F5 and F7 reported that their tribes of origin are related. One branch of this extended family  was recently described [XDH gene by SSCP followed by sequencing revealed a c.1871C>G (p.(S624*)) change and a SNP c.1936A>G in exon 18. Specific RFLP (Nla III) and HRMA assays were developed for detection of the c.1871C>G variant , a 22-year-old female, presented with low back pain and congenital glaucoma and was found to have very low and undetectable levels of UA in the serum and urine, respectively . She dieXDH gene  and was XDH gene in patient IV-15 and the obligatory carriers III-5 and III-6 compared to a control sample, followed by sequencing, revealed a c.141insG (p.(Cys48Lfs*12)) variant and a SNP c.101-35 C>G in IVS 2  and c.1771C>T (p.(Pro591Ser)) variants .Both cases G1 and G2 were Caucasian individuals from Germany. Case G1 was a 46-year-old female with very low levels of UA in the serum and urine ) and c.641del (p.(Pro214Glnfs*4)) variants  of the Fe/S I cluster-binding site, a fully conserved motif in eukaryotes and prokaryotes . Since hthaliana  and expr 150 kDa B with fuUnlike the Cys150Phe variant protein, the yields of the Thr349Ile, Pro591Ser and Arg 776Cys MOCOS variant proteins were variable, though sufficient for investigation of the functional role of the respective amino acid residues in the MOCOS proteins.MOCOS and expressed in E. coli. The Thr349Ile substitution resulted in greatly reduced yields of the variant protein compared to the control  variants, biochemically characterized Arabidopsis thaliana XDH isoform 1 (AtXDH1) was used as a model to study the effect of a substitution of Cys150 in human XDH. Importantly, Cys150 represents the C-terminal Cys residue within a -Cys-Xaa2-Cys-//-Cys-Xaa1-Cys- motif of the Fe/S cluster-binding domain of XDH. Cys150 is predicted to ligate one iron atom of (2Fe-2S) cluster I, as was shown for the corresponding Cys150 in the crystal structure of bovine xanthine dehydrogenase/xanthine oxidase (XDH/XO) [Rhodobacter capsulatus XDH  and has been the subject of biochemical and spectroscopic analyses [Pichia pastoris  . While Canalyses . Moreoveanalyses . For eacanalyses ,37. Thispastoris B, indicaE. coli cysteine desulfurase SufS (PDB no: 1JF9 and 1C0N), Thr349 is located within an α-helix without direct contact with the PLP cofactor. It is thus concluded that the substitution of Thr349 by Ile affects the protein conformation in a way that the protein is less stable compared to the wild-type protein, and the proper insertion and/or stabilization of PLP within the MOCOS backbone is impaired, which, in consequence, is accompanied by a loss of catalytic activity.MOCOS proteins basically consist of two domains: a N-terminal NifS-like domain and a C-terminal Moco-binding domain . The Nifsir3-3 mutant [sir3-3 mutation, i.e., ABA3/Arg723Lys, has shown that the incapacity to activate XDH and AO derives from a reduced ability of the ABA3 variant to bind Moco [5 and cytochrome b5 reductase [Both Pro591 and Arg776 are located within highly conserved motifs of the C-terminal Moco-binding domain of MOCOS B,C. Inte3 mutant  that pre3 mutant . Heterolind Moco . MTARC1 eductase .Within aeductase . It is teductase , so thateductase , Arg776CThe families studied in Israel, with one exception  were characterized, among other features, by a resistance to diet-induced obesity and hepatic steatosis [In view of the diverse pathophysiological roles suggested for the enzymes involved in xanthinuria, the question arises whether the enzymatic deficiencies induce clinically significant perturbances. The proposed pathophysiological role of XDH and its oxidase form XO is highly complex ,53,54. Oteatosis , while ateatosis ,56. The teatosis ,58,59,60teatosis . How canteatosis  was diagteatosis ). Our dateatosis  presenteAn important health aspect of the enzymatic deficiencies in xanthinuria is their involvement in drug metabolisms ,63. One As far as we are aware, we described the largest series of xanthinuria cases reported so far. We reported six novel pathogenic variants, and for the first time, we characterized MOCOS variants underlying type II xanthinuria by in vitro expression studies. We disclosed a new Yemenite-Jewish founder variant and presented molecular and historical data suggesting the prevalence of some variants not only in Israel but, also, elsewhere in the Arab world.